Suzuki Sadafumi, Akamatsu Wado, Kisa Fumihiko, Sone Takefumi, Ishikawa Kei-Ichi, Kuzumaki Naoko, Katayama Hiroyuki, Miyawaki Atsushi, Hattori Nobutaka, Okano Hideyuki
Department of Physiology, Keio University, School of Medicine, Tokyo, Japan.
Center for Genomic and Regenerative Medicine, Juntendo University, School of Medicine, Tokyo, Japan.
Biochem Biophys Res Commun. 2017 Jan 29;483(1):88-93. doi: 10.1016/j.bbrc.2016.12.188. Epub 2017 Jan 3.
Patient-specific induced pluripotent stem cells (iPSCs) show promise for use as tools for in vitro modeling of Parkinson's disease. We sought to improve the efficiency of dopaminergic (DA) neuron induction from iPSCs by the using surface markers expressed in DA progenitors to increase the significance of the phenotypic analysis. By sorting for a CD184/CD44 fraction during neural differentiation, we obtained a population of cells that were enriched in DA neuron precursor cells and achieved higher differentiation efficiencies than those obtained through the same protocol without sorting. This high efficiency method of DA neuronal induction enabled reliable detection of reactive oxygen species (ROS) accumulation and vulnerable phenotypes in PARK2 iPSCs-derived DA neurons. We additionally established a quantitative system using the mt-mKeima reporter system to monitor mitophagy in which mitochondria fuse with lysosomes and, by combining this system with the method of DA neuronal induction described above, determined that mitophagy is impaired in PARK2 neurons. These findings suggest that the efficiency of DA neuron induction is important for the precise detection of cellular phenotypes in modeling Parkinson's disease.
患者特异性诱导多能干细胞（iPSC）有望用作帕金森病体外建模的工具。我们试图通过使用多巴胺能（DA）祖细胞中表达的表面标志物来提高从iPSC诱导DA神经元的效率，以增强表型分析的意义。在神经分化过程中通过分选CD184/CD44组分，我们获得了富含DA神经元前体细胞的细胞群体，并且与未分选的相同方案相比，实现了更高的分化效率。这种高效的DA神经元诱导方法能够可靠地检测PARK2 iPSC衍生的DA神经元中的活性氧（ROS）积累和易损表型。我们还使用线粒体靶向的红色荧光蛋白（mt-mKeima）报告系统建立了一个定量系统来监测线粒体自噬，即线粒体与溶酶体融合，并通过将该系统与上述DA神经元诱导方法相结合，确定PARK2神经元中的线粒体自噬受损。这些发现表明，DA神经元诱导效率对于在帕金森病建模中精确检测细胞表型很重要。
